We propose a transformative approach, inspired by the mechanism of action of antisense oligonucleotides (ASOs), to deliver small molecules that selectively cleave RNA targets in cells and in animals. As a state-of-the- art modality to target RNA, ASOs bind to complementary RNAs and recruit endogenous RNase H, which then cleaves the RNA to eliminate it from the cell. As an alternative to ASOs, we have developed a class of small molecules that selectively bind to and cleave an RNA target and have shown that our new cleaving small molecules are more potent than simple binding compounds. Our approach, dubbed Ribonuclease targeting chimeras (RIBOTACs), engineers small molecules to recruit endogenous RNase L, an RNase expressed at minute levels in cells in a latent form (hence RNase L). The chimeras, comprised of RNA-binding modules and a heterocyclic RNase L-recruiting module, activate RNase L locally at the site of the desired target. We will fully develop our RIBOTAC approach to cleave RNA targets sub-stoichiometrically and catalytically with small molecules, providing a direct means to improve the potency of simple binding compounds. Collectively, we will deliver a platform to program small molecules to cleave specific, malfunctioning RNAs in cells and in animals, with superior properties as compared to ASOs. In support of these goals, we propose in Aim 1 to characterize comprehensively our lead RIBOTAC targeting miR-21 in vitro and in situ, a benchmark for lead optimization. Our new studies show that the binding compound from which this RIBOTAC is derived inhibits metastasis in an orthotopic xenograft model. Further, the RIBOTAC is 20-fold more potent than the simple binding compound in situ for inhibiting miR-21 biogenesis and breast cancer cell phenotypes. Of import, we will study and quantify the selectivity of the RIBOTAC transcriptome- and proteome-wide. In Aim 2, we will lead optimize our RIBOTAC to deliver a proof-of-concept compound with properties amenable for in vivo testing. These DMPK-driven studies will optimize all components of the RIBOTAC, from the RNA-binding modules to the linker that tethers them together to the RNase L-recruiting module. We will rigorously assess top RIBOTACs in the triple negative breast cancer (TNBC) cell line MDA- MB-231, including full assessment and quantification of selectivity transcriptome- and proteome-wide. Finally, in Aim 3, we will study optimized RIBOTACs for activity against a panel of TNBC and patient-derived (PDX) tumor cells ex vivo and in vivo. After confirming miR-21 destruction by our RIBOTACs, we will assess their effects on TNBC cell: (i) proliferation; (ii) survival; (iii) migration and invasion; and (iv) expression of EMT and breast cancer stem cell markers. RIBOTACs with the broadest activity against TNBCs will be evaluated for efficacy in vivo.